Multiple sclerosis (MS) is the result of demyelination in the brain and spinal cord (central nervous system). Symptoms resulting from this demyelination include weakness, visual impairment, incoordination, and paresthesia (abnormal tingling). The course of the disease is largely unpredictable, but often progresses through a cycle of exacerbation of symptoms followed by remission.
Conventional treatments presently employ therapy with ACTH or corticosteroids such as prednisone. Controlled studies suggest that such treatments induce more rapid clearing of acute symptoms and signs but leave the long-term outcome of the disease unaffected. Long-term maintenance therapy with ACTH or corticosteroids is contraindicated. Evidence indicates that immunosuppressant agents have no long-term benefit. (Cecil, Textbook of Medicine, Beeson et al., eds., 15th ed., W. B. Saunders Company, Philadelphia, (1979) page 847)
The etiology of multiple sclerosis is unknown but is linked to a variety of genetic and environmental factors. Both cell-mediated and humoral immune responses, triggered by extraneous or autoantigens may contribute to the pathogenesis of multiple sclerosis. Certain immune response genes may be associated with an increased susceptibility to the disease. The disease may be mediated by T cells that recognize an as yet unidentified autoantigen. For example, experimental allergic encephalomyelitis (EAE), an animal model of demyelinating diseases such as multiple sclerosis, can be induced by immunizing mice with whole myelin or specific myelin components such as myelin basic protein.
In humans with multiple sclerosis, exacerbations are correlated with high levels of neopterin in blood and cerebrospinal fluid. Neopterin is a factor released from monocytes and macrophages in the presence of activated T-cells, thereby implicating these cells as being involved in multiple sclerosis exacerbations. (Fredrickson et al. (1987), Acta Neurol. Scand., 75:352-355; Huber et al. (1984), J. Exp. Med., 160:310-316). At the microscopic level, monocytes, microglial cells (macrophages of the central nervous system), and activated T-cells are found within the demyelinated regions of the nerve cells during multiple sclerosis exacerbations. (Cecil, Textbook of Medicine (1979), Beeson et al. (eds.), W. B. Saunders Co., Philadelphia, Pa.).
Various conventional treatment methodologies have been employed to ameliorate the symptoms of multiple sclerosis. Many of these are directed to use of palliative, anti-inflammatory agents. No treatment to date has had any consistent positive effect on the course of the disease.
Recently, the art has described the use of specific deoxyribosides as anti-inflammatory agents. For instance, U.S. Pat. No. 4,481,197 (Rideout et al.) relates to the use of unsubstituted 3-deaza-2'-deoxyadenosine derivatives in the treatment of inflammation. U.S. Pat. No. 4,381,344 (Rideout et al.) relates to a process for the synthesis of deoxyribosides that utilizes a bacterial phosphorylase.
A deoxyriboside derivative, 2-chloro-2'-deoxyadenosine (CdA), has been found to be an effective agent for the treatment of chronic lymphocytic leukemia and some T cell malignancies. (Carson et al. (1984) Proc. Natl. Acad. Sci. U.S.A., 81:2232-2236; Piro et al. (1988), Blood 72:1069-1073) The pharmacokinetics of orally and subcutaneously administered 2-chloro-2'-deoxyadenosine in the treatment of chronic lymphocytic leukemia have been described and compared. (Liliemark et al. (1992) Journal of Clinical Oncology, 10, (10): 1514-1518; Juliusson et al. (1992) Blood, 80 (Suppl. 1): 1427) Chronic lymphocytic leukemia is a malignancy of B lymphocytes that bear the Leu-1 surface antigen.
The Leu-1 B cells represent a minor proportion of the normal pool of B lymphocytes, usually less than 20 percent. The Leu-1 B cells express surface markers that are typically found on monocytes (Mac-I antigen) and T-lymphocytes (Leu-1 antigen). Approximately 10 percent of patients with chronic lymphocytic leukemia exhibit accompanying autoimmunity, and recently, Leu-1 B cells have been implicated in the pathogenesis of autoimmune diseases.
Phase I clinical trials on human patients with chronic lymphocytic leukemia indicate that infusion of increasing doses of 2-chloro-2'-deoxyadenosine [0.1-0.5 milligrams per kilogram of body weight per day (mg/kg/day)] yielded increasing plasma concentrations of the drug [10-50 nanomolar (nM)]. Those infusions indicated that the drug was well tolerated and did not induce nausea, vomiting or fever. The dose-limiting toxicity was bone marrow suppression, which usually occurred at doses greater than about 0.2 mg/kg/day or at plasma levels of greater than about 20 nM.
Other studies, Montgomery et al. (1959) J. Am. Chem. Soc., 82:463-468, indicated that 2-fluoroadenosine exhibits a relatively high degree of cytotoxicity. Those workers reported that C57 black mice implanted with Adenocarcinoma 755 (Ad755) could tolerate only about 1 milligram per kilogram of body weight. 2-Fluoroadenosine was found to be inactive at that level against Ad755 as well as leukemia L1210 and the Erlich ascites tumor.
U.S. Pat. No. 4,751,221 and its division No. 4,918,179 to Watanabe et al. describe the synthesis and use of several 2-substituted-2'-deoxy-2'-fluoroarabino-furanosyl nucleosides including adenine derivatives. Those compounds were said to have anti-tumor and antitrypanosomal biological activities. Cytotoxicity data showing anti-tumor activity of 2-amino-6-thiopurine, guanine and thiopurine derivatives against murine and human cell lines were reported.
U.S. Pat. No. 5,034,518 to Montgomery et al. teaches the synthesis of 2-substituted-2'-deoxy-2'-fluoroaraadenosines. Those compounds were said to have anticancer activity, and data for prolongation of life of mice transplanted with P388 leukemia cells were provided.
The biochemical activity of 2-CdA in cells has been reviewed by Ernest Beutler. (The Lancet (1992), 340: 952-956--incorporated herein by reference)
The 2',3'-dideoxynucleosides are phosphorylated at the 5'-position in T cells to form the 5'-nucleotide triphosphate derivatives. Those derivatives are well known to be substrates for reverse transcriptase molecules. (Ono et al. (1986) Biochem. Biophys. Res. Comm., 2:498-507)
Those 2',3'-dideoxynucleoside 5'-triphosphates are also utilized by mammalian DNA polymerases beta and gamma. (Waquar et al. (1984) J. Cell. Physiol., 121:402-408) They are, however, poor substrates for DNA polymerase-alpha, the main enzyme responsible for both repair and replicative DNA synthesis in human lymphocytes. In part, these properties may explain the selective anti-HIV activity of the 2',3'dideoxynucleosides.
Chan et al. (1982) J. Cell Physiol., 111:28-32 studied the pathways of pyrimidine nucleotide metabolism in murine peritoneal macrophages and monocytes, and reported undetectable levels of deoxycytidine kinase or thymidine kinase in these cells. High levels of adenosine kinase were found, however.
Similar high levels of adenosine kinase have been found in human monocytes and human monocyte-derived macrophages (MDM). MDM were found to exhibit about one-tenth to about one-fourth the nucleoside kinase activity of GEM T lymphoblasts (e.g. ATCC CCL 119) toward uridine, deoxycytidine and thymidine, and about two-thirds the adenosine kinase activity of GEM cells. In addition, that adenosine kinase activity of MDM cells was at least about 10-fold higher than any of the other kinase activities. Those studies also indicated relatively low levels of nucleoside phosphorylation using AZT, dideoxycytidine (ddC) and 2',3'-dideoxyadenosine (ddA) in intact GEM T lymphoblasts and still lower levels with the MDM.
Several 2-substituted adenosine derivatives have been reported not to be deaminated by adenosine deaminase. For example, Coddington (1965) Biochim. Biophys. Acta, 99:442-451 reported that deoxyadenosine-1-N-oxide, as well as 2-hydroxy-, 2-methyl-, 2-chloro-, 2-acetamido-, and 2-methylthioadenosines were neither substrates nor inhibitors for adenosine deaminase. Montgomery, in Nucleosides, Nucleotides, and Their Biological Applications, Rideout et al. eds., Academic Press, New York, page 19 (1983) provides a table of comparative K.sub.m and V.sub.max data for the deamination of adenosine, 2-haloadenosines 2-halo-deoxyadenosines and 2-fluoroarabinoadenosine that also indicates that those 2-halo adenine derivatives are poor substrates for the enzyme relative to adenine itself. Stoeckler et al. (1982) Biochem. Pharm., 31:1723-1728 reported that the 2'-deoxy-2'-azidoribosyl and 2'-deoxy-2'-azidoarabinosyl-adenine derivatives were substrates for human erythrocytic adenosine deaminase, whereas work of others indicated 2-fluoroadenosine to have negligible activity with adenosine deaminase.
2-Chloro-2'-deoxyadenosine is phosphorylated by non-dividing (normal) human peripheral blood lymphocytes and is converted to the 5'-triphosphate. This adenine derivative is not catabolized significantly by intact human cells or cell extracts, and is phosphorylated efficiently by T lymphocytes. (Carson et al. (1980) Proc. Natl. Acad. Sci. USA, 77:6865-6869)
As discussed before, high levels of adenosine kinase have been found in murine peritoneal macrophages and in human monocytes. Adenosine kinase can phosphorylate 2'-deoxyadenosine derivatives, but does so less efficiently than deoxycytidine kinase. (Hershfield et al. (1982) J. Biol. Chem., 257:6380-6386)
Chemotherapeutic agents are described hereinafter that may be employed as therapeutic agents in the treatment of multiple sclerosis.